The goal of this proposal is to engineer a novel in vitro biomimetic wound healing model to study how human fibroblasts and epithelial cells coordinate tissue closure at the cellular and molecular level. In vivo wound healing is a dynamic morphogenetic process with the goal to close and restore the damaged tissue. A critical stage during tissue closure is re-epithelialization of wounds, a process by which epithelial cells migrate over the denuded wound bed, to restore the barrier. Failure of wounds to re-epithelialize results in chronic wound formation, a condition that affect 6 million Americans annually and carries an estimated cost of US $25 billion per year for the medical system. Hence, understanding the mechanisms that drive re-epithelialization has been a central focus in wound healing research. Due to limitations with animal models, in vitro models have been instrumental to study re-epithelialization by human epithelial cells. Traditional models, such as the scratch wound assay, involve scratching of a monolayer of epithelial cells adherent to a planar substrate, and the time for migrating cells to repopulate the scratch is measured as a proxy for healing. In more advanced co-culture models, the planar substrate is either replaced by a fibroblast-laded collagen hydrogel or by a dermal tissue explant. Whereas these models have a pre-defined substrate as a migration base for epithelial cells, in vivo studies have shown that for full-thickness wounds, the deeper fibrous layers must heal first through the formation of granulation tissue by fibroblasts, before epithelial cells can migrate over this provisional tissue to close the wound. Thus, in in vivo settings, re-epithelialization occurs as fibroblasts deposit a provisional template and reciprocal interactions between fibroblasts an epithelial cells coordinate closure of these two tissue layers. Current in vitro models don't capture this intricate tissue dynamics. Given the dependency of re-epithelialization on the underlying substrate, we hypothesize that fibroblasts mediate the rate of re-epithelialization during wound closure. To address this hypothesis, we propose in aim 1 to build a biomimetic in vitro wound closure model wherein re-epithelialization ensues fibrous tissue repair in wounded engineered microtissues to emulate healing of full thickness wounds. In aim 2, we will use state-of-the art genome editing techniques to elucidate fibroblast- epithelial interactions that regulate fibrous tissue closure and re-epithelialization. These studies will also validate and benchmark our 3D biomimetic model to other wound healing models. In aim 3, we will explore whether fibroblasts from different healthy and pathological tissue sources affect re-epithelialization in our biomimetic model. Ultimately, this project aims to establish a basis for optimizing a wound bed that enables rapid re- epithelialization as a paradigm for promoting tissue regeneration and minimizing scarring.